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Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

Li L, Johnson LA, Dai-Ju JQ, Sandri-Goldin RM - PLoS ONE (2008)

Bottom Line: During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed.Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed.We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA.

ABSTRACT

Background: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/principle findings: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/significance: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

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ICP27 Associates with Hsc70 in vitro.A) GST-binding assays were performed with GST-Hsc70 (1-540) and in vitro translated WT ICP27 and mutants ΔN, D2ΔS5, S5, Δ26-100, H17 and ΔC. Asterisks mark the position of the ICP27 proteins that were seen to bind. Input 35S-labeled proteins are shown in the middle panel. In the bottom panel, a schematic diagram of the 512 amino acid ICP27 coding region, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA binding motif (RGG), arginine-rich region (R2), three predicted KH domains and a zinc-finger-like motif (CCHC) in the C-terminus. The positions of the deletion mutations (dotted lines) are shown below. B) GST binding assays were performed with 35S-labeled, in vitro-translated WT ICP27 and GST-tagged Hsc70 truncated proteins. The arrow shows the position of ICP27. Input GST-Hsc70 proteins are shown in the middle panel. A schematic of the Hsc70 coding region is shown illustrating the nucleotide binding domain (NBD-ATPase), the substrate binding domain (SBD) and the C-terminal domain (CTD).
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pone-0001491-g002: ICP27 Associates with Hsc70 in vitro.A) GST-binding assays were performed with GST-Hsc70 (1-540) and in vitro translated WT ICP27 and mutants ΔN, D2ΔS5, S5, Δ26-100, H17 and ΔC. Asterisks mark the position of the ICP27 proteins that were seen to bind. Input 35S-labeled proteins are shown in the middle panel. In the bottom panel, a schematic diagram of the 512 amino acid ICP27 coding region, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA binding motif (RGG), arginine-rich region (R2), three predicted KH domains and a zinc-finger-like motif (CCHC) in the C-terminus. The positions of the deletion mutations (dotted lines) are shown below. B) GST binding assays were performed with 35S-labeled, in vitro-translated WT ICP27 and GST-tagged Hsc70 truncated proteins. The arrow shows the position of ICP27. Input GST-Hsc70 proteins are shown in the middle panel. A schematic of the Hsc70 coding region is shown illustrating the nucleotide binding domain (NBD-ATPase), the substrate binding domain (SBD) and the C-terminal domain (CTD).

Mentions: ICP27 is a highly interactive protein that has been shown to interact with itself to form multimers [26] and with a number of cellular proteins, including RNAP II [11], [24], splicing factors [27], [28], and mRNA export factors Aly/REF and TAP/NXF1 [29]–[31]. To determine if ICP27 interacts with Hsc70, in vitro binding assays were performed using bacterially-expressed GST-Hsc70 protein encoding amino acids 1 to 540. This includes the Nuclear Binding Domain (NBD- ATPase) and the Substrate Binding Domain (SBD) but not the C-terminal Domain (CTD). In vitro translated, 35S-methionine-labeled wild type and mutant versions of ICP27 were used in these binding assays (Figure 2). WT ICP27 and deletion mutants DΔS5, S5, Δ26-101 and H17 were seen to bind to GST-Hsc70; however, ΔN, in which amino acids 3 to 28 are deleted, and ΔC, in which residues 450 to 512 are deleted, failed to bind to Hsc70 (Figure 2A). These results indicate ICP27 binds Hsc70 in vitro and that both the N- and C-termini of ICP27 must be intact for interaction. A similar result was found for the interaction of ICP27 with RNAP II CTD and with TAP/NXF1 [11], [30], that is, both the N- and C-termini were required for interaction. In assays with Hsc70 truncation mutants, only the 1-540 species of Hsc70 bound to ICP27 (Figure 2B), indicating that the ATPase domain and the substrate binding domain of Hsc70 are required for interaction with ICP27 but not the CTD.


Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

Li L, Johnson LA, Dai-Ju JQ, Sandri-Goldin RM - PLoS ONE (2008)

ICP27 Associates with Hsc70 in vitro.A) GST-binding assays were performed with GST-Hsc70 (1-540) and in vitro translated WT ICP27 and mutants ΔN, D2ΔS5, S5, Δ26-100, H17 and ΔC. Asterisks mark the position of the ICP27 proteins that were seen to bind. Input 35S-labeled proteins are shown in the middle panel. In the bottom panel, a schematic diagram of the 512 amino acid ICP27 coding region, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA binding motif (RGG), arginine-rich region (R2), three predicted KH domains and a zinc-finger-like motif (CCHC) in the C-terminus. The positions of the deletion mutations (dotted lines) are shown below. B) GST binding assays were performed with 35S-labeled, in vitro-translated WT ICP27 and GST-tagged Hsc70 truncated proteins. The arrow shows the position of ICP27. Input GST-Hsc70 proteins are shown in the middle panel. A schematic of the Hsc70 coding region is shown illustrating the nucleotide binding domain (NBD-ATPase), the substrate binding domain (SBD) and the C-terminal domain (CTD).
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Related In: Results  -  Collection

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pone-0001491-g002: ICP27 Associates with Hsc70 in vitro.A) GST-binding assays were performed with GST-Hsc70 (1-540) and in vitro translated WT ICP27 and mutants ΔN, D2ΔS5, S5, Δ26-100, H17 and ΔC. Asterisks mark the position of the ICP27 proteins that were seen to bind. Input 35S-labeled proteins are shown in the middle panel. In the bottom panel, a schematic diagram of the 512 amino acid ICP27 coding region, including the leucine-rich amino terminus (LRR), nuclear localization signal (NLS), RGG box RNA binding motif (RGG), arginine-rich region (R2), three predicted KH domains and a zinc-finger-like motif (CCHC) in the C-terminus. The positions of the deletion mutations (dotted lines) are shown below. B) GST binding assays were performed with 35S-labeled, in vitro-translated WT ICP27 and GST-tagged Hsc70 truncated proteins. The arrow shows the position of ICP27. Input GST-Hsc70 proteins are shown in the middle panel. A schematic of the Hsc70 coding region is shown illustrating the nucleotide binding domain (NBD-ATPase), the substrate binding domain (SBD) and the C-terminal domain (CTD).
Mentions: ICP27 is a highly interactive protein that has been shown to interact with itself to form multimers [26] and with a number of cellular proteins, including RNAP II [11], [24], splicing factors [27], [28], and mRNA export factors Aly/REF and TAP/NXF1 [29]–[31]. To determine if ICP27 interacts with Hsc70, in vitro binding assays were performed using bacterially-expressed GST-Hsc70 protein encoding amino acids 1 to 540. This includes the Nuclear Binding Domain (NBD- ATPase) and the Substrate Binding Domain (SBD) but not the C-terminal Domain (CTD). In vitro translated, 35S-methionine-labeled wild type and mutant versions of ICP27 were used in these binding assays (Figure 2). WT ICP27 and deletion mutants DΔS5, S5, Δ26-101 and H17 were seen to bind to GST-Hsc70; however, ΔN, in which amino acids 3 to 28 are deleted, and ΔC, in which residues 450 to 512 are deleted, failed to bind to Hsc70 (Figure 2A). These results indicate ICP27 binds Hsc70 in vitro and that both the N- and C-termini of ICP27 must be intact for interaction. A similar result was found for the interaction of ICP27 with RNAP II CTD and with TAP/NXF1 [11], [30], that is, both the N- and C-termini were required for interaction. In assays with Hsc70 truncation mutants, only the 1-540 species of Hsc70 bound to ICP27 (Figure 2B), indicating that the ATPase domain and the substrate binding domain of Hsc70 are required for interaction with ICP27 but not the CTD.

Bottom Line: During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed.Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed.We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA.

ABSTRACT

Background: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments.

Methodology/principle findings: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression.

Conclusions/significance: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

Show MeSH
Related in: MedlinePlus